Differential Patterns of Transcriptional Protein Expression May Explain Functional Differences between Hematopoietic Progenitors Derived from Human ESC’s and Fetal Hematopoietic Tissues.

Recent studies suggest that development of human embryoid body-derived hematopoietic progenitor cells (EB-HPC’s) mirrors that of primitive yolk sac-derived hematopoietic progenitors (YS-HPC’s) in their expression of transcription factors known to be critically associated with hematopoietic development. However, the findings of these studies were limited to molecular characterization of heterogeneous EB cultures rather than analysis of hematopoietic lineage-committed cell subsets. We wondered: 1) if the expression patterns were consistent at the protein level in both bulk EB cultures and subsets of hematopoietic-lineage committed cells; and 2) if these expression patterns differed from those found in definitive HPC’s harvested from early fetal hematopoietic tissues. To answer these questions, we compared the intracellular expression of critical transcriptional regulatory proteins from human and rhesus EB-derived HPC’s (hEB-HPC’s and rEB-HPC’s) with hematopoietic progenitors from rhesus fetal liver (rFL-HPC’s) and rhesus placental blood HPC’s (rPB-HPC’s). Rhesus macaque fetal tissue was used in lieu of scarce human fetal tissues. Using multi-color intracellular flow cytometry we correlated the expression of transcriptional proteins (SCL/TAL1, GATA-1, GATA-2, Oct-3/4, HOXB4) with cell surface determinants of hematopoietic commitment (CD34, CD31, CD45) in hEB-HPC’s, rEB-HPC’s, rFL-HPC’s and rPB-HPC’s. Human and rhesus EB16 cells were harvested from identical culture conditions and compared to 0.34 gestation rFL-HPC’s and rPB-HPC’s. The frequency of CD45+ cells was higher in hEB-HPC’s (31%) and rPB-HPC’s (46%) compared to rFL-HPC’s and rEB-HPC’s. A much higher frequency of GATA-1 and HOXB4 expressing cells was seen in both of the fetal cell types when compared to either rhesus or human EB’s. Additionally, the frequency of GATA-2 and SCL expressing cells was comparable in all cell types. Lastly, the expression of Oct3/4 was higher in rPB-HPC’s and rFL-HPC’s when compared to bulk EB cultures. However, analysis of subsets of EB cells expressing hematopoietic antigens (CD45, CD34) revealed co-expression of Oct-3/4 on both CD45+ and CD34+ cell fractions. From these findings, we conclude:

1. critical differences exist in GATA-1 and HOXB4 expression between HPC’s derived from EB cultures and HPC’s harvested during early definitive fetal hematopoietic development; and

2. human EB-derived hematopoietic progenitor cells express persistently high levels of Oct-3/4.

The differences in the expression of these critical transcriptional factors at the protein level may explain the observed functional differences between EB-derived HPC’s and definitive HPC’s from fetal and adult sources.